A new efficient immunoprotocol to detect chromosomal/nuclear proteins along with repetitive DNA in squash...

Protein detection on large somatic chromosomes typically includes paraformaldehyde fixation and squashing of enzymatically softened root tips in a buffer. It often suffers from chromosome clumping, poor chromosome morphology, non-specific fluorescence, insufficient immunoreactivity, which collectively reduce the credibility of immunolabeling, hindering its effective combination with fluorescence in situ hybridization (FISH). Material harvesting and pre-detection steps must be completed within a short time, usually one day, which complicates research. The aim of this study was to develop a simple efficient squash-based protocol for technically demanding formaldehyde-fixed large chromosomes/nuclei (Allium, Scilla, Tradescantia), that ensures: long-term storage of the fixed root tips and of slide preparations, the obtaining of high-quality immunolabeled metaphase plates/nuclear spreads with no or minimal unspecific fluorescence and running a sensitive immunoFISH-karyotyping.

Fixation with 10% buffered formalin was combined with prolonged or overnight storage of the fixed intact tissue in 70% ethanol, digestion with pectinase-cellulase mix in citrate buffer, moderate squashing of root tip tissues in 45% acetic acid, slide freezing followed by ethanol-aided cell adherence to a slide, storage of the preparations in glycerin, one-two cycles of microwave antigen retrieval (MWAR). This resulted in optimal chromosomal/nuclear spreading, good cell adherence to the slide, effective antigen retrieval, reduced/eliminated non-specific fluorescence, good penetration of antibodies. The MWAR-assisted protein redetection could have been performed to strengthen the signals. The protocol was compatible with FISH to perform a sensitive immunoFISH with the rDNA probe and simultaneous visualization of FISH-signals and protein foci.

As a novel approach, the protocol includes an array of steps and options not described in chromosomal immunoprotocols that used aldehyde-fixed root tips for squashing, e.g., fixation with neutral-buffered formalin, storage of root tips in ethanol, squash in acetic acid, MWAR, protein redetection, immunoFISH-aided simultaneous DNA-protein visualization. It ensures chromosomal/nuclear spread of exceptional quality, rapid preparation of the fixing solution, prolonged storage of both fixed tissues and slide preparations, epitope redetection, sensitive immunoFISH-karyotyping. The described methodology provides unprecedented flexibility in laboratory work and significantly expands plant cyto-epigenetic research.